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Follicular atresia during Dacus oleae oogenesis
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Journal of Insect Physiology 52 (2006) 282–290 www.elsevier.com/locate/jinsphys
Follicular atresia during Dacus oleae oogenesis Ioannis P. Nezis, Dimitrios J. Stravopodis, Lukas H. Margaritis, Issidora S. Papassideri Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, 15784 Athens, Greece Received 23 August 2005; received in revised form 2 November 2005; accepted 16 November 2005
Abstract Programmed cell death, constitutes a common fundamental incident that occurs during oogenesis in a variety of different animals. It plays a significant role in the maturation process of the female gamete and also in the removal of abnormal and superfluous cells at certain checkpoints of development. In the present study, we demonstrate the existence of follicular atresia during mid-oogenesis in the olive fruit fly Dacus oleae (Tephritidae). The number of atretic follicles increases following the age of the fly, suggesting for the presence of an age-susceptible process. The atretic follicles contain nurse cells that exhibit chromatin condensation, DNA fragmentation and actin cytoskeleton alterations, as revealed by propidium iodide staining, TUNEL labeling and phalloidin-FITC staining. Conventional light and electron microscopy disclose that the nurse cell remnants are phagocytosed by the adjacent follicle cells. The follicular epithelium also eliminates the oocyte through phagocytosis, resulting to an egg chamber with no compartmentalized organization. The data presented herein are very similar compared to previous reported results in other Diptera species, strongly suggesting the occurrence of a phylogenetically conserved mechanism of follicular atresia. All these observations also support the notion that mid-oogenesis in D. oleae may be the critical regulation point at which superfluous and defective egg chambers are selectively eliminated before they reach maturity. r 2005 Elsevier Ltd. All rights reserved. Keywords: Atresia; Diptera; Follicle cells; Nurse cells; Programmed cell death
1. Introduction Programmed cell death, claims up to 99.9% of the cells in the mammalian female germ line. Approximately 7 million germ cells in the fetal ovaries of humans result in only 400 oocytes in adult life via prenatal and postnatal follicular atresia, a fact that eventually leads to irreversible infertility at the menopause (Tilly, 2001). Although the structure of the follicles in viviparous animals is quite different from oviparous organisms, follicular atresia, which is defined as the degeneration of the follicle, also commonly occurs during oogenesis (specifically for insects see Bell and Bohm, 1975). Oogenesis is a significant biological process that leads to the formation of a highly complex cell, the oocyte. One of its major characteristics is the remarkable gradual increase in the volume of the oocyte, due to accumulation of
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E-mail address: [email protected] (I.S. Papassideri). 0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2005.11.007
nutrients, mRNAs, proteins and organelles required during the early embryonic development. In some insects, including Drosophila melanogaster, the development of the oocyte is supported by a group of cells, termed nurse cells, which are connected to the oocyte and to each other by intercellular bridges, called ring canals (Robinson et al., 1994). The syncytial ensemble of the 15 nurse cells and the oocyte is enveloped by an epithelial monolayer of somatic follicle cells and all together constitute the ovarian follicle, or the egg chamber, which is the structural and functional unit of the ovary (Spradling, 1993; Margaritis and Mazzini, 1998; Trougakos and Margaritis, 2002). The Drosophila ovary consists of a cluster of 18–20 ovarioles, each one representing an independent, developmentally ordered follicle line. According to various morphogenetic criteria, the follicle development has been divided into several distinct stages (14 according to King, 1970; 20 according to Margaritis, 1985, 1986). As it has been previously demonstrated, follicular atresia has been sporadically observed during mid-oogenesis in Drosophila (Giorgi and Deri, 1976; Nezis et al., 2000), or in
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response to nutritional deprivation, ecdysone signaling inhibition, treatment with chemotherapeutic drugs and ectopic death of follicle cells (Buszczak et al., 1999; Chao and Nagoshi, 1999; De Lorenzo et al., 1999; Soller et al., 1999; Nezis et al., 2000; Terashima and Bownes, 2004). The atretic Drosophila follicles contain degenerated nurse cells, mainly characterized by condensed chromatin, fragmented DNA and disorganized actin cytoskeleton (Buszczak et al., 1999; Chao and Nagoshi, 1999; De Lorenzo et al., 1999; Soller et al., 1999; Nezis et al., 2000). Nurse and follicle cell apoptosis is also required for the normal maturation of the developing follicles during the late stages of Drosophila oogenesis (Cavaliere et al., 1998; Foley and Cooley, 1998; Nezis et al., 2000, 2002, for reviews see McCall, 2004; Baum et al., 2005). Our previous results in an another dipteran species, Ceratitis capitata, have shown that follicular atresia also occurs during mid-oogenesis, exhibiting characteristic morphological features of programmed cell death in the nurse cell cluster (Nezis et al., 2003). In the present study, we describe for the first time the intracellular alterations of the atretic follicles that sporadically emerge during midoogenesis in the olive fruit fly Dacus oleae. Degeneration of the D. oleae follicles includes chromatin condensation, DNA fragmentation and actin cytoskeleton organization failure of the nurse cells, as well as absorption of the oocyte and the nurse cell cluster by the surrounding follicle cells. These findings strongly suggest for the presence of a phylogenetically conserved mechanism of follicular atresia in higher Diptera.
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2.3. TUNEL assay Terminal transferase-mediated dUTP nick-end labeling (TUNEL) was performed as follows: ovaries were dissected and separated in individual follicles in Ringer’s solution and fixed in PBS containing 4% formaldehyde plus 0.1% Triton X-100 (Sigma Chemical Co., Germany) for 30 min. After fixation, the samples were rinsed three times and washed twice in PBS for 5 min each. Then, they were incubated with PBS containing 20 mg/ml proteinase K for 10 min and washed three times in PBS for 5 min each. The in situ detection of fragmented genomic DNA was performed with the Boehringer Mannheim TUNEL kit, using fluorescein-labeled dUTP for 3 h at 37 1C in the dark. Following this procedure, the follicles were washed six times in PBS over the course of 1 h and 30 min in the dark, mounted in antifading mounting medium and finally viewed using a Nikon PCM 2000 confocal laser scanning microscope. 2.4. Acridine orange staining Ovaries were dissected in Ringer’s solution and separated into individual follicles. The follicles were then incubated in 1.6 mM acridine orange in Ringer’s solution for 3 min in the dark. Subsequently, they were washed for 5 min in Ringer’s solution and immediately mounted onto vidal glass slides in fresh Ringer’s solution. Elapsed time from dissection to the end of the viewing was restricted to 20 min. Samples were examined using a Nikon PCM 2000 confocal laser scanning microscope.
2. Materials and methods 2.5. Fluorescein-conjugated phalloidin staining 2.1. Dacus oleae culturing D. oleae (Diptera, Tephritidae) adult insects were kept in a 25 1C culture room, fed on standard diet (10% yeast, 40% sugar in water) and slightly etherized before dissection. Dissections were carried out in cold Ringer’s solution and ovaries were separated into single ovarioles. Staging of the developing egg chambers during oogenesis was accomplished according to Margaritis (1985, 1986) and Nezis et al. (2001).
Ovaries were fixed in PBS containing 4% formaldehyde for 30 min and subsequently permeabilized for 25 min in PBS containing 4% formaldehyde plus 0.1% Triton X-100. After three washes in PBS for 5 min each, the follicles were stained in PBS containing 1 mg/ml fluorescein-conjugated phalloidin (Sigma Chemical Co., Germany) for 2 h in the dark. Finally, the samples were rinsed three times with PBS, washed in PBS for 15 min, mounted in antifading mounting medium and viewed using a Nikon PCM 2000 confocal laser scanning microscope.
2.2. Propidium iodide staining Ovaries were dissected in Ringer’s solution, fixed in PBS containing 4% formaldehyde for 30 min and subsequently permeabilized for 25 min in PBS containing 4% formaldehyde plus 0.1% Triton X-100. After three washes in PBS for 5 min each, the follicles were incubated with 600 mg/ml RNaseA in PBS for 1 h and stained with 700 ng/ml propidium iodide in PBS for 15 min. Finally, the samples were mounted in 90% glycerol containing 1.4-diazabicyclo (2.2.2) octane (Sigma Chemical Co., Germany) to avoid fading and viewed using a Nikon PCM 2000 confocal laser scanning microscope.
2.6. Conventional light and transmission electron microscopy D. oleae follicles were processed for conventional light and transmission electron microscopy as described elsewhere (Margaritis et al., 1980; Nezis et al., 2005). Sections (1 mm thick) were stained with 0.5% toluidine blue in 1% sodium borate and observed with an Olympus BH-2 light microscope. Ultrathin sections were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate and subsequently observed in a Philips EM 300 electron microscope.
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3. Results 3.1. Follicular atresia in Dacus oleae ovary We used the TUNEL labeling approach to detect the stage-specific atretic follicles in whole mount preparations of D. oleae ovaries. As it is shown in Table 1, in young (3-days old) olive fruit flies we can observe at least 3 atretic follicles in each ovary (number of ovaries tested n ¼ 48). Each atretic egg chamber contains apoptotic nurse cell
Table 1 Mean number of atretic follicles (identified by TUNEL assay) in Dacus oleae ovaries Mean number of atretic follicles per ovary
Ovaries tested
3-days-old fly: 3.2 25-days-old fly: 10.5
48 42
The number of atretic follicles increases significantly (43 ) by the age of the fly.
Fig. 1. Morphological characteristic features of normal and atretic follicles (stage 8) during mid-oogenesis in Dacus oleae. Normal follicles: (a), (c), (e), (g). Atretic follicles: (b), (d), (f), (h). (a, b) Confocal micrographs of propidium iodide stained follicles. The chromatin of the nurse cell nuclei of the atretic follicle is condensed (b, arrows) compared to the one of the normal follicle (a). (c, d) Confocal micrographs after TUNEL labeling. The nurse cell nuclei of the atretic follicle contain fragmented DNA (d, arrows). No signal can be detected in the normal follicle (c). (e, f) Confocal micrographs after acridine orange staining. The nurse cell nuclei of the atretic follicle are characterized by fragmented DNA (f, arrows). No signal is evident in the normal follicle (e). (g, h) Confocal micrographs of phalloidin-FITC stained egg chambers. Note the actin cytoskeleton organization failure in the atretic follicle (h) compared to the normal one (g). Arrow in (h): unusual actin-rich structure. FN: follicle cell nucleus, NN: nurse cell nucleus, OC: oocyte, FC: follicle cell, NC: nurse cell, RC: ring canal. Bars: 50 mm.
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nuclei mainly characterized by fragmented genomic DNA (Fig. 1d). Interestingly, the number of atretic follicles increases significantly following the age of the fly (25-days old) and results in, at least, 10 follicles per ovary (number of ovaries tested n ¼ 42) (Table 1). The atretic egg chambers also exhibit TUNEL-positive intense signals in their nurse cell nuclei (data not shown). 3.2. Morphology of atretic follicles in Dacus oleae Follicular atresia in D. oleae occurs exclusively during mid-oogenesis (stage 8) (Fig. 1). We applied several cytochemical methods to demonstrate the morphology of these degenerated egg chambers. First, we used propidium iodide staining to detect chromatin condensation events. As it is clearly illustrated (Fig. 1b), the nurse cell nuclei of the atretic follicles are characterized by highly condensed chromatin compared to the normal egg chambers of the same stage (Fig. 1a). On the other hand, the follicle cell nuclei do not seem to contain condensed chromatin and their morphology looks normal (Figs. 1a and b). We next applied the TUNEL assay in order to detect DNA fragmentation events. As it is shown in the case of the normal egg chambers during mid-oogenesis (Fig. 1c), we were not able to observe any positive signal for DNA fragmentation in all the cell types of the tested follicles (nurse cells, follicle cells and oocyte). On the contrary, the
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atretic ones were characterized by nurse cell nuclei that reacted strongly with TUNEL reagents, indicating that they all contained highly fragmented DNA (Fig. 1d). To further confirm the presence of DNA fragmentation incidents, we selected another widely used marker of DNA fragmentation, named acridine orange. As it is shown (Fig. 1f), we could detect distinct positive signals in the nurse cell nuclei of the atretic egg chambers. No signal was observed in the follicle cell layer and the oocyte (Fig. 1f). Additionally, as expected, there were no positive reactions developed in all the tested normal follicles during mid-oogenesis (Fig. 1e). Since the actin cytoskeleton alterations constitute a hallmark of programmed cell death, we decided to use the phalloidin-FITC staining approach to visualize the actin cytoskeleton organization and arrangement in D. oleae follicles during mid-oogenesis. A stage 8 normal egg chamber is characterized by the following structural pattern: in the nurse cells, the F-actin is localized subcortically and in the numerous ring canals, as well (Fig. 1g). In the oocyte, a strong fluorescent signal is subcortically distributed and can be also detected in the follicle cells (Fig. 1g). On the other hand, the atretic follicles display actin cytoskeleton organization failure in the nurse cell and oocyte compartments and only the follicle cells exhibit the regular staining pattern (Fig. 1h). In the nurse cell compartment, a few unusual actin-rich
Fig. 2. Light micrographs of normal and atretic egg chambers during Dacus oleae mid-oogenesis. (a) Light micrograph of a stage 8 normal egg chamber. The follicle cells (FC), the nurse cells (NN: nurse cell nuclei) and the oocyte (OC) exhibit a well-defined organization and distribution. (b) Light micrograph of an atretic egg chamber at the initial phase of degeneration. The nurse cell nuclei (NN) are condensed, while the follicle cells (FC) contain large vacuoles with condensed material (arrows). (c) Light micrograph of an atretic egg chamber at the last phase of degeneration. The oocyte and nurse cell cluster have lost their compartmentalized arrangement and structure. The follicular epithelium contains condensed cell bodies (arrows). Large vacuoles are clearly detected in the oocyte area (small arrow). (d) High resolution light micrograph of the same atretic egg chamber illustrated in Fig. 2b. The follicular epithelium contains numerous phagosomes filled with condensed material highly resembling the yolk spheres (arrows). Some yolk spheres exhibit degenerated morphology and fragmented structure (small arrows). FCN: follicle cell nucleus, OC: oocyte, YS: yolk spheres. Bars: 50 mm.
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structures can be also detected (Fig. 1h). It is important to underline that the morphology of the atretic follicles remains the same regardless of the age of the fly. 3.3. Ultrastructural morphology of the atretic follicles: a loss of the compartmentalized integrity We first used the conventional light microscopy approach to demonstrate the cellular structure and organization of D. oleae follicles during mid-oogenesis. The nurse cell cluster of a stage 8 normal follicle contains almost spherical euchromatic nuclei with several electrondense nucleoli (Figs. 2a and 3b). The follicular epithelium and oocyte are properly ordered and regularly arranged (Figs. 2a, 3a and b). In the initial phase of atresia, the chromatin of the nurse cell nuclei starts to condense, while the follicle cells activate the oocyte phagocytosis process (Figs. 2b and d). In this phase of degeneration, the follicular epithelium surrounding the oocyte efficiently phagocytoses the ooplasm and the yolk spheres (Figs. 2d, 3c, e and f). Interestingly, the yolk spheres appear to have obtained an unusual structure, compared to the ones of a normal egg chamber (Figs. 3a and h). They exhibit unusual holes, being also fragmented in small pieces (Figs. 2d and 3h). On the other hand, the follicular epithelium surrounding the nurse cells successfully phagocytoses the nurse cell cytoplasm (Figs. 3d and g). These follicles cell subpopulations contain large phagosomes filled with vacuoles and condensed cytoplasmic material, resembling rough endoplasmic reticulum profiles in a degeneration process (Fig. 3g). The nuclei of the nurse cells, during this initial phase of degeneration, appear to have obtained condensed chromatin, while the nuclear membrane has acquired a ruptured structure (Fig. 3i). During the last phase of degeneration, the atretic follicles lose their cellular compartmentalized organization and integrity. More specifically, the oocyte and nurse cell cluster can not be distinguished from each other, while the follicular epithelium is misplaced, containing numerous electron-dense
bodies (Fig. 2c). Detailed ultrastructural analysis revealed that the follicle cell layer is mainly characterized by the presence of numerous large phagosomes (Figs. 4a–d) with heterogeneous, regarding their content, internal morphology (Fig. 4a). As it is illustrated (Figs. 4b–d), they have engulfed nuclear and cytoplasmic apoptotic material, mainly identified as condensed chromatin, vacuoles, multi-layered membranes and swollen mitochondria with loss of their cristae internal structures (Figs. 4c and d). 4. Discussion As we have previously demonstrated, programmed cell death occurs physiologically in D. oleae nurse cells cluster during the late stages of oogenesis and is essential for the normal development and maturation of the egg chamber (Nezis et al., 2001). This process is characterized by chromatin condensation, DNA fragmentation, actin cytoskeleton reorganization and phagocytosis of the apoptotic remnants by the adjacent follicle cells (Nezis et al., 2001). In the present study, we describe another distinct pattern of programmed cell death that occurs sporadically during D. oleae oogenesis, named follicular atresia. Atresia of D. oleae follicles is detected exclusively during mid-oogenesis. All the atretic follicles contain nurse cells that exhibit chromatin condensation, DNA fragmentation and actin cytoskeleton organization failure. The nurse cell remnants are phagocytosed by the adjacent follicular epithelium. The follicle cells also eliminate the oocyte through phagocytosis, resulting to an egg chamber without any compartmentalized organization. The regulation of mid-oogenesis cell death in Drosophila is mainly controlled by the proper balance between juvenile hormone and ecdysone (Soller et al., 1999; Buszczak et al., 1999). Interestingly, the C. capitata homologous member of the ecdysone receptor, CcEcR, is expressed at its highest levels during mid-oogenesis (Verras et al., 2002). Additionally, in mosquitoes there is a significant increase in the levels of ecdysone following a blood meal, which allows for the
Fig. 3. Ultrastructural morphology of normal and atretic egg chambers during the initial phase of degeneration. (a) Electron micrograph of a stage 8 normal follicle illustrating the follicular epithelium (FC) surrounding the oocyte (OC) that contains the characteristic yolk spheres (YS). No phagosomes can be detected in these follicle cell subpopulations. (b) Electron micrograph of a stage 8 normal follicle illustrating the follicular epithelium (FC) surrounding the nurse cells (NC). The nurse cell nucleus (NCN) is mainly euchromatic, exhibiting a well-organized structure. No phagosomes can be detected in these follicle cell subpopulations. (c) Electron micrograph of an atretic egg chamber (stage 8) at the initial phase of degeneration. The follicular epithelium surrounding the oocyte contains large phagosomes (arrows) filled with cytoplasmic material (small arrows). FCN: follicle cell nucleus. The two squared areas are, respectively, illustrated in higher magnification in (e) and (f), intending to better clarify and characterize the nature of the engulfed material. The engulfed material demonstrated in the left squared area [also magnified in (e)] is a fragmented part of a yolk sphere. The ultrastructural texture and morphology of this formation appear very similar with the ones of degenerating yolk spheres presented in (h) (inset). (f) Illustrates the right squared area in high resolution, where condensed, likely cytoplasmic, masses can be also detected. (d) Electron micrograph of an atretic egg chamber (stage 8) at the initial phase of degeneration. The follicular epithelium surrounding the nurse cell cluster contains large phagosomes (arrow) filled with cytoplasmic material. FCN: follicle cell nucleus, NC: nurse cell. (g) High resolution micrograph of the phagosome shown in (d). The phagosome contains numerous vacuoles and condensed cytoplasmic material similar to the one illustrated in (c) and (f). (h) The yolk spheres in the atretic egg chambers have obtained a degenerated morphology, exhibiting unusual holes and multi-fragmented internal structures (arrows). The squared area is shown in higher magnification as an inset [notice the similar texture of the squared material in between (c {e}) and (h {inset})]. (i) The nurse cell nucleus (NCN) of a representative atretic egg chamber at the initial phase of degeneration, contains condensed chromatin, while the nuclear membrane exhibits a unique morphology of a ruptured structure, mainly characterized by numerous gaps and breaks all around the nuclear periphery (arrows). Notice the invasion of cytoplasmic vacuoles into the nuclear area, due to the loss of nuclear membrane integrity. Bars: (a–d) 10; (e and f) 0.1; (g) 1; (h) 1; (h, inset) 0.1 and (i) 5 mm.
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Fig. 4. Ultrastructural morphology of representative atretic egg chambers, during the last phase of degeneration. (a) Electron micrograph of an atretic egg chamber (stage 8) at the last phase of degeneration. The follicular epithelium contains large phagosomes of heterogeneous composition and morphology (arrows). FCN: follicle cells nuclei. (b) High resolution micrograph of a phagosome shown in (a). This particular phagosome contains distinct nuclear (big arrow) and cytoplasmic (small arrow) material. The texture and nature (as judged by comparative morphology) of the phagocytosed nuclear material appear very similar to the nurse cell nuclei ones. A portion of a degenerated nurse cell nucleus is illustrated in high resolution as an inset. The cytoplasmic material seems to contain multi-layered membrane structures. (c) Electron micrograph of an atretic egg chamber (stage 8) at the last phase of degeneration. A follicle cell has already engulfed a phagosome filled with cytoplasmic material (arrow). FCN: follicle cell nucleus. (d) High resolution micrograph of (c). The cytoplasmic material consists of numerous vacuoles, multi-layered membranes and swollen mitochondria with loss of their cristae internal foldings (arrows). Bars: a: 10, b and d: 1 and c: 5 mm.
onset of vitellogenesis (Raikhel et al., 2002). Thus, it is very likely that ecdysone and juvenile hormone levels can also control whether an egg chamber survives or perishes during D. oleae mid-oogenesis. In Drosophila, the levels of juvenile hormone in young female flies are substantially higher than in mature flies (Bownes and Rembold, 1987; Altaratz et al., 1991). We assume that the different relative balance of ecdysone and juvenile hormone between young and old flies could be associated with the increase in the number of the atretic follicles in old D. oleae flies. Recent studies in Drosophila have shown that caspases and their inhibitors play a significant role in the execution of the mid-oogenesis cell death. Over-expression of an active form of the effector caspase Dcp-1 in the ovary results in the degeneration of egg chambers during midoogenesis (Peterson et al., 2003). This phenotype is also
characterized by high levels of the active caspase Drice and can be suppressed by over-expression of the apoptotic inhibitor DIAP-1 (Peterson et al., 2003). Additionally, flies defective for Dcp-1 display a lack of sporadically degenerated stage 7–8 egg chambers (Laundrie et al., 2003; Nezis et al., 2005). So, it is reasonable to speculate that the D. oleae caspase homologous member of Dcp-1 is responsible for the execution of the cell death program characterizing the follicular atresia process. Interestingly, homologous caspase family members have been shown to exist in other Diptera species, like the mosquito Anopheles stephensi and C. capitata (Abraham et al., 2004; Nezis et al., unpublished). In atretic follicles of the mosquito Culex pipiens pallens, ovarian cathepsin-like proteinases, which have been accumulated within the developing oocyte primarily for
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embryogenesis, are activated to digest yolk proteins and other cellular substrate structures (Uchida et al., 2001). Furthermore, the follicle cells incorporate yolk granules from the oocyte (Uchida et al., 2004). The above observation is very similar to the one we describe herein concerning the oocyte elimination in D. oleae atretic follicles. Thus, we could speculate that the degeneration of the oocyte is regulated by a stage-specific activation of distinct endogenous ovarian proteinases. Despite our efforts, we were not able to elucidate how the nucleus of the oocyte degenerates in atretic follicles during midoogenesis. Since it has been previously reported that the oocyte death likely progresses very rapidly (Reynaud and Driancourt, 2000), we might have simply missed picking up a degenerated oocyte nucleus in our sample preparations. In all the atretic egg chambers tested, we could not detect any characteristic feature of cell death in the epithelial follicle cells. This observation significantly differs from previously reported data in mosquitoes (Hopwood et al., 2001; Uchida et al., 2004), where the follicular epithelium in the atretic egg chambers degenerates during the last phase of atresia. Follicular atresia in mosquitoes becomes a massive process after a blood meal, or plasmodium infection (Hopwood et al., 2001; Uchida et al., 2004), since the accurate regulation of the total number of the follicles is really urgent. So, we assume that the follicle cell layer death program promotes for a more effective egg chamber atresia in mosquitoes. It is possible that our observations concerning the resistance of the follicle cells to the stagespecific cell death during mid-oogenesis could be directly attributed to certain differences in the physiology between mosquitoes and fruit flies. However, we can not exclude the possibility that follicle cells of atretic egg chambers in D. oleae degenerate at an ensuing phase during oogenesis, being efficiently absorbed at the entry of the lateral oviducts by epithelial cells, as it has been previously reported for Drosophila melanogaster (Nezis et al., 2002). The availability of food and other environmental factors can influence the competence of Drosophila egg chambers to develop beyond stage 8 (Spradling, 1993; DrummondBarbosa and Spradling, 2001; Terashima and Bownes, 2004). Furthermore, disruption of the follicle cell layer induces germ cell death during mid-oogenesis in Drosophila (De Lorenzo et al., 1999; Chao and Nagoshi, 1999). Moreover, in vitro treatment of Drosophila egg chambers with the apoptotic inducers etoposide and staurosporine triggers nurse cell apoptosis during stages 7–8 (Nezis et al., 2000). When an egg chamber enters vitellogenesis during stage 8 it needs a significant amount of resources. Therefore, a pre-vitellogenic checkpoint may serve to remove superfluous, or defective, egg chambers and thus may control the total number of available egg chambers for ovulation and prevent the waste of precious nutrients. Consequently, mid-oogenesis in D. oleae could be considered as the critical regulation point at which superfluous and defective egg chambers are selectively eliminated. Thus, it is very likely that this phenomenon constitutes an
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essential protective mechanism throughout D. oleae oogenesis that results in the programmed cell death of superfluous, spontaneously mutated, defective and abnormal follicles before they reach maturity. The morphological features of follicular atresia we describe in the present study are very similar to the ones occurring in other higher Diptera species, such as Drosophila melanogaster (Giorgi and Deri, 1976; Nezis et al., 2000) and C. capitata (Nezis et al., 2003). All the above findings strongly suggest for the presence of a phylogenetically conserved mechanism of atresia in higher Diptera during oogenesis. Acknowledgements The authors would like to thank Professor H.M. Moutsopoulos (Department of Pathophysiology, Medical School, University of Athens, Athens, Greece) for kindly providing confocal laser scanning microscope facilities. This work was supported by a grant to I.S. Papassideri (Pythagoras II). References Abraham, E.G., Islam, S., Srinivasan, P., Ghosh, A.K., Valenzuela, J.G., Ribeiro, J.M., Kafatos, F.C., Dimopoulos, G., Jacobs-Lorena, M., 2004. Analysis of the Plasmodium and Anopheles transcriptional repertoire during ookinete development and midgut invasion. Journal of Biological Chemistry 279, 5573–5580. Altaratz, M., Applebaum, S.W., Richard, D.S., Gilbert, L.I., Segal, D., 1991. Regulation of juvenile hormone synthesis in wild-type and apterous mutant Drosophila. Molecular and Cellular Endocrinology 81, 205–216. Baum, J.S., St. George, J.P., McCall, K., 2005. Programmed cell death in the germline. Seminars in Cell and Developmental Biology 16, 245–259. Bell, W.J., Bohm, M.K., 1975. Oosorption in insects. Biological Review 50, 373–396. Bownes, M., Rembold, H., 1987. The titre of juvenile hormone during the pupal and adult stages of the life cycle of Drosophila melanogaster. European Journal of Biochemistry 164, 709–712. Buszczak, M., Freeman, M.R., Carlson, J.R., Bender, M., Cooley, L., Segraves, W.A., 1999. Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila. Development 126, 4581–4589. Cavaliere, V., Taddei, C., Gargiulo, G., 1998. Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster. Development, Genes and Evolution 208, 106–112. Chao, S., Nagoshi, R.N., 1999. Induction of apoptosis in the germline and follicle layer of Drosophila egg chambers. Mechanisms of Development 88, 159–172. De Lorenzo, C., Strand, D., Mechler, B.M., 1999. Requirement of Drosophila l(2)gl function for survival of the germline cells and organization of the follicle cells in a columnar epithelium during oogenesis. International Journal of Developmental Biology 43, 207–217. Drummond-Barbosa, D., Spradling, A.C., 2001. Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Developmental Biology 231, 265–278. Foley, K., Cooley, L., 1998. Apoptosis in late stage Drosophila nurse cells does not require genes within the H99 deficiency. Development 125, 1075–1082.
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Nezis, I.P., Stravopodis, D.J., Papassideri, I.S., Stergiopoulos, C., Margaritis, L.H., 2005. Morphological irregularities and features of resistance to apoptosis in the dcp-1/pita double mutated egg chambers during Drosophila oogenesis. Cell Motility and the Cytoskeleton 60, 14–23. Peterson, J.S., Barkett, M., McCall, K., 2003. Stage-specific regulation of caspase activity in Drosophila oogenesis. Developmental Biology 260, 113–123. Raikhel, A.S., Kokoza, V.A., Zhu, J., Martin, D., Wang, S.F., Li, C., Sun, G., Ahmed, A., Dittmer, N., Attardo, G., 2002. Molecular biology of mosquito vitellogenesis: from basic studies to genetic engineering of antipathogen immunity. Insect Biochemistry and Molecular Biology 32, 1275–1286. Reynaud, K., Driancourt, M.A., 2000. Oocyte atrition. Molecular and Cellular Endocrinology 163, 101–108. Robinson, D.N., Cant, K., Cooley, L., 1994. Morphogenesis of Drosophila ovarian ring canals. Development 120, 2015–2025. Soller, M., Bownes, M., Kubli, E., 1999. Control of oocyte maturation in sexually mature Drosophila females. Developmental Biology 208, 337–351. Spradling, A.C., 1993. Developmental genetics of oogenesis. In: Bate, M., Martinez-Arias, A. (Eds.), The Development of Drosophila melanogaster, vol. 1. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 1–70. Terashima, J., Bownes, M., 2004. Translating available food into the number of eggs laid by Drosophila melanogaster. Genetics 167, 1711–1719. Tilly, J.L., 2001. Commuting the death sentence: how oocytes strive to survive. Nature Reviews in Molecular Cell Biology 2, 838–848. Trougakos, I.P., Margaritis, L.H., 2002. Novel morphological and physiological aspects of insect eggs. In: Hilker, M., Meiners, T. (Eds.), Chemoecology of Insect Eggs and Egg Deposition. Blackwell Wissenschaftsverlag, Berlin, Germany, pp. 3–36. Uchida, K., Ohmori, D., Ueno, T., Nishizuka, M., Eshita, Y., Fukunaga, A., Kominami, E., 2001. Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens. Developmental Biology 237, 68–78. Uchida, K., Nishizuka, M., Ohmori, D., Ueno, T., Eshita, Y., Fukunaga, A., 2004. Follicular epithelial cell apoptosis of atretic follicles within developing ovaries of the mosquito Culex pipiens pallens. Journal of Insect Physiology 50, 903–912. Verras, M., Gourzi, P., Zacharopoulou, A., Mintzas, A.C., 2002. Developmental profiles and ecdysone regulation of the mRNAs for two ecdysone receptor isoforms in the Mediterranean fruit fly Ceratitis capitata. Insect Molecular Biology 11, 553–565.
Journal of Insect Physiology 52 (2006) 282–290 www.elsevier.com/locate/jinsphys
Follicular atresia during Dacus oleae oogenesis Ioannis P. Nezis, Dimitrios J. Stravopodis, Lukas H. Margaritis, Issidora S. Papassideri Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, 15784 Athens, Greece Received 23 August 2005; received in revised form 2 November 2005; accepted 16 November 2005
Abstract Programmed cell death, constitutes a common fundamental incident that occurs during oogenesis in a variety of different animals. It plays a significant role in the maturation process of the female gamete and also in the removal of abnormal and superfluous cells at certain checkpoints of development. In the present study, we demonstrate the existence of follicular atresia during mid-oogenesis in the olive fruit fly Dacus oleae (Tephritidae). The number of atretic follicles increases following the age of the fly, suggesting for the presence of an age-susceptible process. The atretic follicles contain nurse cells that exhibit chromatin condensation, DNA fragmentation and actin cytoskeleton alterations, as revealed by propidium iodide staining, TUNEL labeling and phalloidin-FITC staining. Conventional light and electron microscopy disclose that the nurse cell remnants are phagocytosed by the adjacent follicle cells. The follicular epithelium also eliminates the oocyte through phagocytosis, resulting to an egg chamber with no compartmentalized organization. The data presented herein are very similar compared to previous reported results in other Diptera species, strongly suggesting the occurrence of a phylogenetically conserved mechanism of follicular atresia. All these observations also support the notion that mid-oogenesis in D. oleae may be the critical regulation point at which superfluous and defective egg chambers are selectively eliminated before they reach maturity. r 2005 Elsevier Ltd. All rights reserved. Keywords: Atresia; Diptera; Follicle cells; Nurse cells; Programmed cell death
1. Introduction Programmed cell death, claims up to 99.9% of the cells in the mammalian female germ line. Approximately 7 million germ cells in the fetal ovaries of humans result in only 400 oocytes in adult life via prenatal and postnatal follicular atresia, a fact that eventually leads to irreversible infertility at the menopause (Tilly, 2001). Although the structure of the follicles in viviparous animals is quite different from oviparous organisms, follicular atresia, which is defined as the degeneration of the follicle, also commonly occurs during oogenesis (specifically for insects see Bell and Bohm, 1975). Oogenesis is a significant biological process that leads to the formation of a highly complex cell, the oocyte. One of its major characteristics is the remarkable gradual increase in the volume of the oocyte, due to accumulation of
Corresponding author. Tel.: +30 210 7274546; fax: +30 210 7274742.
E-mail address: [email protected] (I.S. Papassideri). 0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2005.11.007
nutrients, mRNAs, proteins and organelles required during the early embryonic development. In some insects, including Drosophila melanogaster, the development of the oocyte is supported by a group of cells, termed nurse cells, which are connected to the oocyte and to each other by intercellular bridges, called ring canals (Robinson et al., 1994). The syncytial ensemble of the 15 nurse cells and the oocyte is enveloped by an epithelial monolayer of somatic follicle cells and all together constitute the ovarian follicle, or the egg chamber, which is the structural and functional unit of the ovary (Spradling, 1993; Margaritis and Mazzini, 1998; Trougakos and Margaritis, 2002). The Drosophila ovary consists of a cluster of 18–20 ovarioles, each one representing an independent, developmentally ordered follicle line. According to various morphogenetic criteria, the follicle development has been divided into several distinct stages (14 according to King, 1970; 20 according to Margaritis, 1985, 1986). As it has been previously demonstrated, follicular atresia has been sporadically observed during mid-oogenesis in Drosophila (Giorgi and Deri, 1976; Nezis et al., 2000), or in
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response to nutritional deprivation, ecdysone signaling inhibition, treatment with chemotherapeutic drugs and ectopic death of follicle cells (Buszczak et al., 1999; Chao and Nagoshi, 1999; De Lorenzo et al., 1999; Soller et al., 1999; Nezis et al., 2000; Terashima and Bownes, 2004). The atretic Drosophila follicles contain degenerated nurse cells, mainly characterized by condensed chromatin, fragmented DNA and disorganized actin cytoskeleton (Buszczak et al., 1999; Chao and Nagoshi, 1999; De Lorenzo et al., 1999; Soller et al., 1999; Nezis et al., 2000). Nurse and follicle cell apoptosis is also required for the normal maturation of the developing follicles during the late stages of Drosophila oogenesis (Cavaliere et al., 1998; Foley and Cooley, 1998; Nezis et al., 2000, 2002, for reviews see McCall, 2004; Baum et al., 2005). Our previous results in an another dipteran species, Ceratitis capitata, have shown that follicular atresia also occurs during mid-oogenesis, exhibiting characteristic morphological features of programmed cell death in the nurse cell cluster (Nezis et al., 2003). In the present study, we describe for the first time the intracellular alterations of the atretic follicles that sporadically emerge during midoogenesis in the olive fruit fly Dacus oleae. Degeneration of the D. oleae follicles includes chromatin condensation, DNA fragmentation and actin cytoskeleton organization failure of the nurse cells, as well as absorption of the oocyte and the nurse cell cluster by the surrounding follicle cells. These findings strongly suggest for the presence of a phylogenetically conserved mechanism of follicular atresia in higher Diptera.
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2.3. TUNEL assay Terminal transferase-mediated dUTP nick-end labeling (TUNEL) was performed as follows: ovaries were dissected and separated in individual follicles in Ringer’s solution and fixed in PBS containing 4% formaldehyde plus 0.1% Triton X-100 (Sigma Chemical Co., Germany) for 30 min. After fixation, the samples were rinsed three times and washed twice in PBS for 5 min each. Then, they were incubated with PBS containing 20 mg/ml proteinase K for 10 min and washed three times in PBS for 5 min each. The in situ detection of fragmented genomic DNA was performed with the Boehringer Mannheim TUNEL kit, using fluorescein-labeled dUTP for 3 h at 37 1C in the dark. Following this procedure, the follicles were washed six times in PBS over the course of 1 h and 30 min in the dark, mounted in antifading mounting medium and finally viewed using a Nikon PCM 2000 confocal laser scanning microscope. 2.4. Acridine orange staining Ovaries were dissected in Ringer’s solution and separated into individual follicles. The follicles were then incubated in 1.6 mM acridine orange in Ringer’s solution for 3 min in the dark. Subsequently, they were washed for 5 min in Ringer’s solution and immediately mounted onto vidal glass slides in fresh Ringer’s solution. Elapsed time from dissection to the end of the viewing was restricted to 20 min. Samples were examined using a Nikon PCM 2000 confocal laser scanning microscope.
2. Materials and methods 2.5. Fluorescein-conjugated phalloidin staining 2.1. Dacus oleae culturing D. oleae (Diptera, Tephritidae) adult insects were kept in a 25 1C culture room, fed on standard diet (10% yeast, 40% sugar in water) and slightly etherized before dissection. Dissections were carried out in cold Ringer’s solution and ovaries were separated into single ovarioles. Staging of the developing egg chambers during oogenesis was accomplished according to Margaritis (1985, 1986) and Nezis et al. (2001).
Ovaries were fixed in PBS containing 4% formaldehyde for 30 min and subsequently permeabilized for 25 min in PBS containing 4% formaldehyde plus 0.1% Triton X-100. After three washes in PBS for 5 min each, the follicles were stained in PBS containing 1 mg/ml fluorescein-conjugated phalloidin (Sigma Chemical Co., Germany) for 2 h in the dark. Finally, the samples were rinsed three times with PBS, washed in PBS for 15 min, mounted in antifading mounting medium and viewed using a Nikon PCM 2000 confocal laser scanning microscope.
2.2. Propidium iodide staining Ovaries were dissected in Ringer’s solution, fixed in PBS containing 4% formaldehyde for 30 min and subsequently permeabilized for 25 min in PBS containing 4% formaldehyde plus 0.1% Triton X-100. After three washes in PBS for 5 min each, the follicles were incubated with 600 mg/ml RNaseA in PBS for 1 h and stained with 700 ng/ml propidium iodide in PBS for 15 min. Finally, the samples were mounted in 90% glycerol containing 1.4-diazabicyclo (2.2.2) octane (Sigma Chemical Co., Germany) to avoid fading and viewed using a Nikon PCM 2000 confocal laser scanning microscope.
2.6. Conventional light and transmission electron microscopy D. oleae follicles were processed for conventional light and transmission electron microscopy as described elsewhere (Margaritis et al., 1980; Nezis et al., 2005). Sections (1 mm thick) were stained with 0.5% toluidine blue in 1% sodium borate and observed with an Olympus BH-2 light microscope. Ultrathin sections were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate and subsequently observed in a Philips EM 300 electron microscope.
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3. Results 3.1. Follicular atresia in Dacus oleae ovary We used the TUNEL labeling approach to detect the stage-specific atretic follicles in whole mount preparations of D. oleae ovaries. As it is shown in Table 1, in young (3-days old) olive fruit flies we can observe at least 3 atretic follicles in each ovary (number of ovaries tested n ¼ 48). Each atretic egg chamber contains apoptotic nurse cell
Table 1 Mean number of atretic follicles (identified by TUNEL assay) in Dacus oleae ovaries Mean number of atretic follicles per ovary
Ovaries tested
3-days-old fly: 3.2 25-days-old fly: 10.5
48 42
The number of atretic follicles increases significantly (43 ) by the age of the fly.
Fig. 1. Morphological characteristic features of normal and atretic follicles (stage 8) during mid-oogenesis in Dacus oleae. Normal follicles: (a), (c), (e), (g). Atretic follicles: (b), (d), (f), (h). (a, b) Confocal micrographs of propidium iodide stained follicles. The chromatin of the nurse cell nuclei of the atretic follicle is condensed (b, arrows) compared to the one of the normal follicle (a). (c, d) Confocal micrographs after TUNEL labeling. The nurse cell nuclei of the atretic follicle contain fragmented DNA (d, arrows). No signal can be detected in the normal follicle (c). (e, f) Confocal micrographs after acridine orange staining. The nurse cell nuclei of the atretic follicle are characterized by fragmented DNA (f, arrows). No signal is evident in the normal follicle (e). (g, h) Confocal micrographs of phalloidin-FITC stained egg chambers. Note the actin cytoskeleton organization failure in the atretic follicle (h) compared to the normal one (g). Arrow in (h): unusual actin-rich structure. FN: follicle cell nucleus, NN: nurse cell nucleus, OC: oocyte, FC: follicle cell, NC: nurse cell, RC: ring canal. Bars: 50 mm.
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nuclei mainly characterized by fragmented genomic DNA (Fig. 1d). Interestingly, the number of atretic follicles increases significantly following the age of the fly (25-days old) and results in, at least, 10 follicles per ovary (number of ovaries tested n ¼ 42) (Table 1). The atretic egg chambers also exhibit TUNEL-positive intense signals in their nurse cell nuclei (data not shown). 3.2. Morphology of atretic follicles in Dacus oleae Follicular atresia in D. oleae occurs exclusively during mid-oogenesis (stage 8) (Fig. 1). We applied several cytochemical methods to demonstrate the morphology of these degenerated egg chambers. First, we used propidium iodide staining to detect chromatin condensation events. As it is clearly illustrated (Fig. 1b), the nurse cell nuclei of the atretic follicles are characterized by highly condensed chromatin compared to the normal egg chambers of the same stage (Fig. 1a). On the other hand, the follicle cell nuclei do not seem to contain condensed chromatin and their morphology looks normal (Figs. 1a and b). We next applied the TUNEL assay in order to detect DNA fragmentation events. As it is shown in the case of the normal egg chambers during mid-oogenesis (Fig. 1c), we were not able to observe any positive signal for DNA fragmentation in all the cell types of the tested follicles (nurse cells, follicle cells and oocyte). On the contrary, the
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atretic ones were characterized by nurse cell nuclei that reacted strongly with TUNEL reagents, indicating that they all contained highly fragmented DNA (Fig. 1d). To further confirm the presence of DNA fragmentation incidents, we selected another widely used marker of DNA fragmentation, named acridine orange. As it is shown (Fig. 1f), we could detect distinct positive signals in the nurse cell nuclei of the atretic egg chambers. No signal was observed in the follicle cell layer and the oocyte (Fig. 1f). Additionally, as expected, there were no positive reactions developed in all the tested normal follicles during mid-oogenesis (Fig. 1e). Since the actin cytoskeleton alterations constitute a hallmark of programmed cell death, we decided to use the phalloidin-FITC staining approach to visualize the actin cytoskeleton organization and arrangement in D. oleae follicles during mid-oogenesis. A stage 8 normal egg chamber is characterized by the following structural pattern: in the nurse cells, the F-actin is localized subcortically and in the numerous ring canals, as well (Fig. 1g). In the oocyte, a strong fluorescent signal is subcortically distributed and can be also detected in the follicle cells (Fig. 1g). On the other hand, the atretic follicles display actin cytoskeleton organization failure in the nurse cell and oocyte compartments and only the follicle cells exhibit the regular staining pattern (Fig. 1h). In the nurse cell compartment, a few unusual actin-rich
Fig. 2. Light micrographs of normal and atretic egg chambers during Dacus oleae mid-oogenesis. (a) Light micrograph of a stage 8 normal egg chamber. The follicle cells (FC), the nurse cells (NN: nurse cell nuclei) and the oocyte (OC) exhibit a well-defined organization and distribution. (b) Light micrograph of an atretic egg chamber at the initial phase of degeneration. The nurse cell nuclei (NN) are condensed, while the follicle cells (FC) contain large vacuoles with condensed material (arrows). (c) Light micrograph of an atretic egg chamber at the last phase of degeneration. The oocyte and nurse cell cluster have lost their compartmentalized arrangement and structure. The follicular epithelium contains condensed cell bodies (arrows). Large vacuoles are clearly detected in the oocyte area (small arrow). (d) High resolution light micrograph of the same atretic egg chamber illustrated in Fig. 2b. The follicular epithelium contains numerous phagosomes filled with condensed material highly resembling the yolk spheres (arrows). Some yolk spheres exhibit degenerated morphology and fragmented structure (small arrows). FCN: follicle cell nucleus, OC: oocyte, YS: yolk spheres. Bars: 50 mm.
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structures can be also detected (Fig. 1h). It is important to underline that the morphology of the atretic follicles remains the same regardless of the age of the fly. 3.3. Ultrastructural morphology of the atretic follicles: a loss of the compartmentalized integrity We first used the conventional light microscopy approach to demonstrate the cellular structure and organization of D. oleae follicles during mid-oogenesis. The nurse cell cluster of a stage 8 normal follicle contains almost spherical euchromatic nuclei with several electrondense nucleoli (Figs. 2a and 3b). The follicular epithelium and oocyte are properly ordered and regularly arranged (Figs. 2a, 3a and b). In the initial phase of atresia, the chromatin of the nurse cell nuclei starts to condense, while the follicle cells activate the oocyte phagocytosis process (Figs. 2b and d). In this phase of degeneration, the follicular epithelium surrounding the oocyte efficiently phagocytoses the ooplasm and the yolk spheres (Figs. 2d, 3c, e and f). Interestingly, the yolk spheres appear to have obtained an unusual structure, compared to the ones of a normal egg chamber (Figs. 3a and h). They exhibit unusual holes, being also fragmented in small pieces (Figs. 2d and 3h). On the other hand, the follicular epithelium surrounding the nurse cells successfully phagocytoses the nurse cell cytoplasm (Figs. 3d and g). These follicles cell subpopulations contain large phagosomes filled with vacuoles and condensed cytoplasmic material, resembling rough endoplasmic reticulum profiles in a degeneration process (Fig. 3g). The nuclei of the nurse cells, during this initial phase of degeneration, appear to have obtained condensed chromatin, while the nuclear membrane has acquired a ruptured structure (Fig. 3i). During the last phase of degeneration, the atretic follicles lose their cellular compartmentalized organization and integrity. More specifically, the oocyte and nurse cell cluster can not be distinguished from each other, while the follicular epithelium is misplaced, containing numerous electron-dense
bodies (Fig. 2c). Detailed ultrastructural analysis revealed that the follicle cell layer is mainly characterized by the presence of numerous large phagosomes (Figs. 4a–d) with heterogeneous, regarding their content, internal morphology (Fig. 4a). As it is illustrated (Figs. 4b–d), they have engulfed nuclear and cytoplasmic apoptotic material, mainly identified as condensed chromatin, vacuoles, multi-layered membranes and swollen mitochondria with loss of their cristae internal structures (Figs. 4c and d). 4. Discussion As we have previously demonstrated, programmed cell death occurs physiologically in D. oleae nurse cells cluster during the late stages of oogenesis and is essential for the normal development and maturation of the egg chamber (Nezis et al., 2001). This process is characterized by chromatin condensation, DNA fragmentation, actin cytoskeleton reorganization and phagocytosis of the apoptotic remnants by the adjacent follicle cells (Nezis et al., 2001). In the present study, we describe another distinct pattern of programmed cell death that occurs sporadically during D. oleae oogenesis, named follicular atresia. Atresia of D. oleae follicles is detected exclusively during mid-oogenesis. All the atretic follicles contain nurse cells that exhibit chromatin condensation, DNA fragmentation and actin cytoskeleton organization failure. The nurse cell remnants are phagocytosed by the adjacent follicular epithelium. The follicle cells also eliminate the oocyte through phagocytosis, resulting to an egg chamber without any compartmentalized organization. The regulation of mid-oogenesis cell death in Drosophila is mainly controlled by the proper balance between juvenile hormone and ecdysone (Soller et al., 1999; Buszczak et al., 1999). Interestingly, the C. capitata homologous member of the ecdysone receptor, CcEcR, is expressed at its highest levels during mid-oogenesis (Verras et al., 2002). Additionally, in mosquitoes there is a significant increase in the levels of ecdysone following a blood meal, which allows for the
Fig. 3. Ultrastructural morphology of normal and atretic egg chambers during the initial phase of degeneration. (a) Electron micrograph of a stage 8 normal follicle illustrating the follicular epithelium (FC) surrounding the oocyte (OC) that contains the characteristic yolk spheres (YS). No phagosomes can be detected in these follicle cell subpopulations. (b) Electron micrograph of a stage 8 normal follicle illustrating the follicular epithelium (FC) surrounding the nurse cells (NC). The nurse cell nucleus (NCN) is mainly euchromatic, exhibiting a well-organized structure. No phagosomes can be detected in these follicle cell subpopulations. (c) Electron micrograph of an atretic egg chamber (stage 8) at the initial phase of degeneration. The follicular epithelium surrounding the oocyte contains large phagosomes (arrows) filled with cytoplasmic material (small arrows). FCN: follicle cell nucleus. The two squared areas are, respectively, illustrated in higher magnification in (e) and (f), intending to better clarify and characterize the nature of the engulfed material. The engulfed material demonstrated in the left squared area [also magnified in (e)] is a fragmented part of a yolk sphere. The ultrastructural texture and morphology of this formation appear very similar with the ones of degenerating yolk spheres presented in (h) (inset). (f) Illustrates the right squared area in high resolution, where condensed, likely cytoplasmic, masses can be also detected. (d) Electron micrograph of an atretic egg chamber (stage 8) at the initial phase of degeneration. The follicular epithelium surrounding the nurse cell cluster contains large phagosomes (arrow) filled with cytoplasmic material. FCN: follicle cell nucleus, NC: nurse cell. (g) High resolution micrograph of the phagosome shown in (d). The phagosome contains numerous vacuoles and condensed cytoplasmic material similar to the one illustrated in (c) and (f). (h) The yolk spheres in the atretic egg chambers have obtained a degenerated morphology, exhibiting unusual holes and multi-fragmented internal structures (arrows). The squared area is shown in higher magnification as an inset [notice the similar texture of the squared material in between (c {e}) and (h {inset})]. (i) The nurse cell nucleus (NCN) of a representative atretic egg chamber at the initial phase of degeneration, contains condensed chromatin, while the nuclear membrane exhibits a unique morphology of a ruptured structure, mainly characterized by numerous gaps and breaks all around the nuclear periphery (arrows). Notice the invasion of cytoplasmic vacuoles into the nuclear area, due to the loss of nuclear membrane integrity. Bars: (a–d) 10; (e and f) 0.1; (g) 1; (h) 1; (h, inset) 0.1 and (i) 5 mm.
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Fig. 4. Ultrastructural morphology of representative atretic egg chambers, during the last phase of degeneration. (a) Electron micrograph of an atretic egg chamber (stage 8) at the last phase of degeneration. The follicular epithelium contains large phagosomes of heterogeneous composition and morphology (arrows). FCN: follicle cells nuclei. (b) High resolution micrograph of a phagosome shown in (a). This particular phagosome contains distinct nuclear (big arrow) and cytoplasmic (small arrow) material. The texture and nature (as judged by comparative morphology) of the phagocytosed nuclear material appear very similar to the nurse cell nuclei ones. A portion of a degenerated nurse cell nucleus is illustrated in high resolution as an inset. The cytoplasmic material seems to contain multi-layered membrane structures. (c) Electron micrograph of an atretic egg chamber (stage 8) at the last phase of degeneration. A follicle cell has already engulfed a phagosome filled with cytoplasmic material (arrow). FCN: follicle cell nucleus. (d) High resolution micrograph of (c). The cytoplasmic material consists of numerous vacuoles, multi-layered membranes and swollen mitochondria with loss of their cristae internal foldings (arrows). Bars: a: 10, b and d: 1 and c: 5 mm.
onset of vitellogenesis (Raikhel et al., 2002). Thus, it is very likely that ecdysone and juvenile hormone levels can also control whether an egg chamber survives or perishes during D. oleae mid-oogenesis. In Drosophila, the levels of juvenile hormone in young female flies are substantially higher than in mature flies (Bownes and Rembold, 1987; Altaratz et al., 1991). We assume that the different relative balance of ecdysone and juvenile hormone between young and old flies could be associated with the increase in the number of the atretic follicles in old D. oleae flies. Recent studies in Drosophila have shown that caspases and their inhibitors play a significant role in the execution of the mid-oogenesis cell death. Over-expression of an active form of the effector caspase Dcp-1 in the ovary results in the degeneration of egg chambers during midoogenesis (Peterson et al., 2003). This phenotype is also
characterized by high levels of the active caspase Drice and can be suppressed by over-expression of the apoptotic inhibitor DIAP-1 (Peterson et al., 2003). Additionally, flies defective for Dcp-1 display a lack of sporadically degenerated stage 7–8 egg chambers (Laundrie et al., 2003; Nezis et al., 2005). So, it is reasonable to speculate that the D. oleae caspase homologous member of Dcp-1 is responsible for the execution of the cell death program characterizing the follicular atresia process. Interestingly, homologous caspase family members have been shown to exist in other Diptera species, like the mosquito Anopheles stephensi and C. capitata (Abraham et al., 2004; Nezis et al., unpublished). In atretic follicles of the mosquito Culex pipiens pallens, ovarian cathepsin-like proteinases, which have been accumulated within the developing oocyte primarily for
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embryogenesis, are activated to digest yolk proteins and other cellular substrate structures (Uchida et al., 2001). Furthermore, the follicle cells incorporate yolk granules from the oocyte (Uchida et al., 2004). The above observation is very similar to the one we describe herein concerning the oocyte elimination in D. oleae atretic follicles. Thus, we could speculate that the degeneration of the oocyte is regulated by a stage-specific activation of distinct endogenous ovarian proteinases. Despite our efforts, we were not able to elucidate how the nucleus of the oocyte degenerates in atretic follicles during midoogenesis. Since it has been previously reported that the oocyte death likely progresses very rapidly (Reynaud and Driancourt, 2000), we might have simply missed picking up a degenerated oocyte nucleus in our sample preparations. In all the atretic egg chambers tested, we could not detect any characteristic feature of cell death in the epithelial follicle cells. This observation significantly differs from previously reported data in mosquitoes (Hopwood et al., 2001; Uchida et al., 2004), where the follicular epithelium in the atretic egg chambers degenerates during the last phase of atresia. Follicular atresia in mosquitoes becomes a massive process after a blood meal, or plasmodium infection (Hopwood et al., 2001; Uchida et al., 2004), since the accurate regulation of the total number of the follicles is really urgent. So, we assume that the follicle cell layer death program promotes for a more effective egg chamber atresia in mosquitoes. It is possible that our observations concerning the resistance of the follicle cells to the stagespecific cell death during mid-oogenesis could be directly attributed to certain differences in the physiology between mosquitoes and fruit flies. However, we can not exclude the possibility that follicle cells of atretic egg chambers in D. oleae degenerate at an ensuing phase during oogenesis, being efficiently absorbed at the entry of the lateral oviducts by epithelial cells, as it has been previously reported for Drosophila melanogaster (Nezis et al., 2002). The availability of food and other environmental factors can influence the competence of Drosophila egg chambers to develop beyond stage 8 (Spradling, 1993; DrummondBarbosa and Spradling, 2001; Terashima and Bownes, 2004). Furthermore, disruption of the follicle cell layer induces germ cell death during mid-oogenesis in Drosophila (De Lorenzo et al., 1999; Chao and Nagoshi, 1999). Moreover, in vitro treatment of Drosophila egg chambers with the apoptotic inducers etoposide and staurosporine triggers nurse cell apoptosis during stages 7–8 (Nezis et al., 2000). When an egg chamber enters vitellogenesis during stage 8 it needs a significant amount of resources. Therefore, a pre-vitellogenic checkpoint may serve to remove superfluous, or defective, egg chambers and thus may control the total number of available egg chambers for ovulation and prevent the waste of precious nutrients. Consequently, mid-oogenesis in D. oleae could be considered as the critical regulation point at which superfluous and defective egg chambers are selectively eliminated. Thus, it is very likely that this phenomenon constitutes an
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essential protective mechanism throughout D. oleae oogenesis that results in the programmed cell death of superfluous, spontaneously mutated, defective and abnormal follicles before they reach maturity. The morphological features of follicular atresia we describe in the present study are very similar to the ones occurring in other higher Diptera species, such as Drosophila melanogaster (Giorgi and Deri, 1976; Nezis et al., 2000) and C. capitata (Nezis et al., 2003). All the above findings strongly suggest for the presence of a phylogenetically conserved mechanism of atresia in higher Diptera during oogenesis. Acknowledgements The authors would like to thank Professor H.M. Moutsopoulos (Department of Pathophysiology, Medical School, University of Athens, Athens, Greece) for kindly providing confocal laser scanning microscope facilities. This work was supported by a grant to I.S. Papassideri (Pythagoras II). References Abraham, E.G., Islam, S., Srinivasan, P., Ghosh, A.K., Valenzuela, J.G., Ribeiro, J.M., Kafatos, F.C., Dimopoulos, G., Jacobs-Lorena, M., 2004. Analysis of the Plasmodium and Anopheles transcriptional repertoire during ookinete development and midgut invasion. Journal of Biological Chemistry 279, 5573–5580. Altaratz, M., Applebaum, S.W., Richard, D.S., Gilbert, L.I., Segal, D., 1991. Regulation of juvenile hormone synthesis in wild-type and apterous mutant Drosophila. Molecular and Cellular Endocrinology 81, 205–216. Baum, J.S., St. George, J.P., McCall, K., 2005. Programmed cell death in the germline. Seminars in Cell and Developmental Biology 16, 245–259. Bell, W.J., Bohm, M.K., 1975. Oosorption in insects. Biological Review 50, 373–396. Bownes, M., Rembold, H., 1987. The titre of juvenile hormone during the pupal and adult stages of the life cycle of Drosophila melanogaster. European Journal of Biochemistry 164, 709–712. Buszczak, M., Freeman, M.R., Carlson, J.R., Bender, M., Cooley, L., Segraves, W.A., 1999. Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila. Development 126, 4581–4589. Cavaliere, V., Taddei, C., Gargiulo, G., 1998. Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster. Development, Genes and Evolution 208, 106–112. Chao, S., Nagoshi, R.N., 1999. Induction of apoptosis in the germline and follicle layer of Drosophila egg chambers. Mechanisms of Development 88, 159–172. De Lorenzo, C., Strand, D., Mechler, B.M., 1999. Requirement of Drosophila l(2)gl function for survival of the germline cells and organization of the follicle cells in a columnar epithelium during oogenesis. International Journal of Developmental Biology 43, 207–217. Drummond-Barbosa, D., Spradling, A.C., 2001. Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Developmental Biology 231, 265–278. Foley, K., Cooley, L., 1998. Apoptosis in late stage Drosophila nurse cells does not require genes within the H99 deficiency. Development 125, 1075–1082.
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